5324 |Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 This journal is ©the Owner Societies 2016
Cite this: Phys.Chem.Chem.Phys.,
2016, 18,5324
Concentration dependent effects of urea binding
to poly(N-isopropylacrylamide) brushes: a
combined experimental and numerical study†
Samantha Micciulla,
a
Julian Michalowsky,
b
Martin A. Schroer,
cd
Christian Holm,
b
Regine von Klitzing
a
and Jens Smiatek*
b
The binding effects of osmolytes on the conformational behavior of grafted polymers are studied in this
work. In particular, we focus on the interactions between urea and poly(N-isopropylacrylamide)
(PNIPAM) brushes by monitoring the ellipsometric brush thickness for varying urea concentrations over a
broad temperature range. The interpretation of the obtained data is supported by atomistic molecular
dynamics simulations, which provide detailed insights into the experimentally observed concentration-
dependent effects on PNIPAM–urea interaction. In particular, in the low concentration regime (c
u
r
0.5 mol L
1
) a preferential exclusion of urea from PNIPAM chains is observed, while in the high concen-
tration regime (2 rc
u
r7 mol L
1
) a preferential binding of the osmolyte to the polymer surface is
found. In both regimes, the volume phase transition temperature (T
tr
) decreases with increasing urea
concentration. This phenomenon derives from two different effects depending on urea concentration:
(i) for c
u
r0.5 mol L
1
, the decrease of T
tr
is explained by a decrease of the chemical potential of
bulk water in the surrounding aqueous phase; (ii) for c
u
Z2 mol L
1
, the lower T
tr
is explained by the
favorable replacement of water molecules by urea, which can be regarded as a cross-linker between
adjacent PNIPAM chains. Significant effects of the concentration-dependent urea binding on the brush
conformation are noticed: at c
u
= 0.5 mol L
1
, although urea is loosely embedded between the hydrated
polymer chains, it enhances the brush swelling by excluded volume effects. Beyond 0.5 mol L
1
, the
stronger interaction between PNIPAM and urea reduces the chain hydration, which in combination with
cross-linking of monomer units induces the shrinkage of the polymer brush.
1 Introduction
Osmolytes are low-weight organic molecules which contribute
to the regulation of the osmotic pressure inside the cells. Their
presence allows biological cells to counteract a change of the
external osmotic pressure in such a way that the isotonic balance
is ensured. In addition, osmolytes have a strong influence on the
structural conformation of proteins.
1–4
It was reported that in
high concentrated solutions of denaturants like guanidinium
chloride or urea, protein unfolding occurs when a critical osmolyte
concentration is reached.
5–9
In contrast, the stabilization of protein
structures was validated for protectants like trimethylamine-N-
oxide (TMAO) and hydroxyectoine.
10–13
Experimental findings further evidenced that low concentra-
tions of urea and guanidinium chloride can even stabilize
protein native structures
9
and lead to the fluidization of lipid
bilayers and monolayers.
12–14
The mechanisms and the different
accumulation properties
15–18
for protectants and denaturants
around molecular surfaces can be brought into relation with
the ‘law of matching water affinities’.
15,19,20
As it was also recently
demonstrated by computer simulations, the resulting binding
behavior is a consequence of a subtle interplay between enthalpic
and entropic effects, and the underlying charge of the solvated
surface.
21
In addition, further simulations of thermoresponsive
polymers in presence of different salt concentration also reveal
a concentration dependent change of binding properties for
chaotropic salts.
22
The possibility of investigating these effects
is reasoned by the fact that the binding or the exclusion mecha-
nism of the co-solute has a crucial influence on the stability of the
macromolecule and therefore on the corresponding transition
temperatures. Two distinct osmolyte binding mechanisms have
been proposed: (1) direct interactions, related to the binding of
the osmolyte to the surface of the macromolecule via hydrogen
a
Stranski-Laboratorium, Institut fu
¨r Chemie, Technische Universita
¨t Berlin,
D-10623 Berlin, Germany
b
Institut fu
¨r Computerphysik, Universita
¨t Stuttgart, D-70569 Stuttgart, Germany.
E-mail: smiatek@icp.uni-stuttgart.de
c
Deutsches Elektronen-Synchrotron DESY, D-22607 Hamburg, Germany
d
The Hamburg Centre for Ultrafast Imaging (CUI), D-22761 Hamburg, Germany
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cp07544k
Received 7th December 2015,
Accepted 21st January 2016
DOI: 10.1039/c5cp07544k
www.rsc.org/pccp
PCCP
PAPER
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
View Journal
| View Issue
This journal is ©the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 | 5325
bonds, salt bridges or dispersion interactions,
23–27
and (2) indirect
interactions, mostly imposed by the influence of the osmolyte on
the local water shell around the surface.
28,29
However, it is still
under debate which mechanism is predominant.
Due to the chemical variety and structural complexity,
simplified homopolymers are more preferred than proteins in
fundamental studies. Among them, poly(N-isopropylacrylamide)
(PNIPAM) received a lot of scientific attention, because the
mechanism underlying its thermoresponsive behavior, namely
a coil-to-globule transition above the lower critical solution
temperature (LCST), is closely related to the loss of protein
structure upon addition of denaturating agents and resembles
the behavior found for the cold denaturation of proteins.
30
It
has been validated that the predominant effects displayed
above the LCST rely on the low number of hydrating water
molecules bound to the macromolecule.
31–35
Hence, the analysis
of hydration properties, molecular size and transition tempera-
tures represents a valid tool to study the influence of added
solutes on the temperature-induced conformational changes of
PNIPAM.
36,37
In particular for PNIPAM, recent studies found that highly
concentrated urea solutions lead to a significant decrease of the
LCST.
38
From the results of Fourier transformed infrared
spectroscopy experiments, it was concluded that mainly direct
interactions between urea and the carbonyl group of NIPAM
promote the collapse into a globular state via a stabilizing
bivalent hydrogen bond. In addition, more recent numerical
studies also indicated that entropic effects arising from the
accumulation of urea clouds around the PNIPAM molecule might
have a crucial influence on the thermal behavior.
27,39,40
Typical studies of PNIPAM–urea interactions were performed
in bulk
41–44
and very rarely for osmolyte concentrations as low
as 0.5 mol L
1
. Indeed, grafted chains are the base of a large
number of smart coatings,
45–47
therefore their conformational
behavior in presence of osmolytes is of high interest. In this work,
for the first time a systematic investigation of the swelling and
collapse properties of PNIPAM brushes over a broad range of urea
concentration from 0.1 to 7 mol L
1
is presented. The experi-
mental approach consisted in monitoring the optical thickness of
PNIPAM brushes by ellipsometry as a function of temperature
for urea concentrations between 0.1 and 7 mol L
1
.Fromthe
obtained data, the transition temperature (T
tr
) and the swelling
degree (f
sw
) of PNIPAM below (288 K) and above (328 K) the
critical solution temperature were calculated. The hydration
properties with regard to the underlying polymer–urea inter-
actions were deduced from the observed swelling and collapse
behavior. The interpretation of the experimental data is validated
by atomistic MD simulation of PNIPAM chains in two different
concentration regimes (low and high molar concentration) and
for temperatures below and above the phase transition. The
numerical studies further allow us to elucidate the balance
between solvent (water) and co-solute (urea) binding on the
conformational equilibrium of the polymer chain for the different
combinations of urea concentration and temperature. In particular,
at low molar urea concentrations, the observed shift of the
transition temperature was governed by the dilution effect of
urea on bulk water, which changes its chemical potential from
pure solvent to dilute solution, and therefore favors brush
dehydration across the phase transition. This effect was due
to a preferential exclusion of urea from the macromolecule. In
contrast, at high molar urea concentrations, the preferential
binding of urea to PNIPAM was responsible for the pronounced
decrease of T
tr
, which was reasoned by the weakening of
PNIPAM–water interactions, and the cross-linking of the osmolyte
between adjacent PNIPAM chains.
Our study offers a deeper understanding on the impact of
direct urea binding and indirect effects on the conformational
equilibrium of the polymer. It contributes to widen the pre-
vious knowledge in this field and to understand the behavior of
proteins in presence of osmolytes.
2 Experimental section
2.1 Materials
Silicon wafers (Siltronic AG, Germany) were used as solid substrates
forthepolymerbrushes.N-Isopropylacrylamide (NIPAM) (98%,
stabilized by methylhydroquinone) was purchased from TCI Tokyo
Chemical Industry; N,N,N0,N00,N00-pentamethyldiethylene-triamine
(PMDETA), Cu(I)Cl,methanolandureawereallpurchasedfrom
Sigma Aldrich (Germany). All reagents were used as received
without any further purification.
2.2 Synthesis of polymer brushes
Polymer brushes were grown from silicon wafers by Atom
Transfer Radical Polymerization (ATRP).
48
First of all, a self-
assembled monolayer of the ATRP initiator 2-bromo-2-methyl-
N-[3-(triethoxysilyl)-propyl]-propanamide (BTPAm)
49
in dry toluene
(4 mL/10 mL) was adsorbed on the substrates for 24 h. A polymer-
ization mixture of 2.83 g NIPAM in 50 mL of methanol and water
(1 : 1 v/v), 183 mLN,N,N0,N00,N00-pentamethyldiethylene-triamine
(PMDETA) and 25 mg copper(I) chloride was prepared and
degassed by N
2
bubbling for 40 minutes. The initiator-modified
substrates were soaked in the reaction mixture and the polymer-
ization was run for 8 minutes, followed by termination in water/
methanol mixture and thorough rinsing in Milli-Q water. The
grafting density swas calculated considering the molecular
structure of the initiator and the measured thickness of the
self-assembled monolayer (details are reported in the ESI†). A
value of sB0.4 nm
2
was estimated, which corresponds to a
moderate brush regime
50
and allows urea to access the full
solvent accessible surface area of PNIPAM. The brush thickness
of the dry state (1.9 0.1% r.h.) was measured by ellipsometry
after drying the samples in a sealed cell flushed with nitrogen
stream for 20 minutes. The measured value was d
dry
= (40.5
0.9) nm and it remained constant for at least 10 minutes.
The molecular weight (M
W
) of PNIPAM chains was estimated
from the grafting density sand the dry thickness d
dry
,according
to the relation
49,51,52
MW¼rddryNA
s(1)
Paper PCCP
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
5326 |Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 This journal is ©the Owner Societies 2016
with rthe polymer mass density (1.1 g mL
1
)andN
A
the Avogadro
number. The obtained value for M
W
was E67 000 g mol
1
(ca.
590 monomers per chain). Reported polydispersity indices (PDI)
for ATRP polymerization are mostly between 1.2 and 1.5.
53,54
A low PDI is also expected for our system, given the low standard
deviation of the brush thickness (ca. 0.8% of the brush thickness).
The swollen thicknesses of PNIPAM brushes in solutions of urea
at different concentrations varied between 100 and 150 nm at
288 K, and between 47 and 80 nm at 328 K. The values are
reported in Table S1 of the ESI.†
2.3 Ellipsometry
Ellipsometry is a non-destructive, optical technique which is
based on the detection of the change of polarization of light
upon reflection from a substrate. Such a change is described by
two parameters, cand D, which are related to the amplitude (E)
and the phase (d) of light by the following relations:
tan c¼
Er
p
.Ei
p
Er
s
Ei
s
(2)
D=(d
r
p
d
r
s
)(d
i
p
d
i
s
) (3)
where the subscripts p, s indicate the parallel and perpendi-
cular components of the light, while the superscripts i, r specify
the incoming and reflected beam. The reflectivity properties
of a sample can be expressed by the reflection coefficients,
r
p
and r
s
,
rp¼
Er
p
Ei
p
eidr
pdi
p
ðÞ (4)
rs¼Er
s
Ei
s
eidr
sdi
s
ðÞ (5)
whose combination with eqn (2) and (3) yield the fundamental
equation of ellipsometry:
tan ceiD¼rp
rs
:(6)
The use of a proper layer model, which describes the system
under measurement (substrate-film-medium) in terms of its
optical properties (thickness dand refractive index n) can be
obtained from the measured parameters Dand c, which in turn
describe the interaction between polarized light and sample.
Specific literature is available on the topic to which the reader is
addressed for a more detailed description of the technique.
55,56
For the data analysis, the measured values of Dand Cwere
fitted with a five box model consisting, from the bottom to the
top, of (i) silicon substrate (n= 3.8850, d=N), (ii) silicon oxide
(n= 1.46, d= 1.5 nm), (iii) initiator BTPAm monolayer (n= 1.50,
d= 0.7 nm), (iv) polymer brushes (n=n
brush
,d=d
brush
), (v)
liquid medium (n
calc
,d=N). From the measured values of
Dand c, the corresponding combination of n=n
brush
,d=d
brush
for the polymer brushes is obtained. The refractive index of
solutions containing different urea concentrations was estimated
by the empirical relations elaborated by Warren and Gordon
57
from experimental data on urea solutions. The corresponding
values are reported in Table S2 of the ESI.†The degree of swelling
f
sw
for PNIPAM brushes was calculated from the thickness d
dry
of the dry brush and the thickness d
sw
(T) of the swollen brush at a
given temperature Tby
fsw ¼dswðTÞddry
ddry
100½%:(7)
The brush collapse from the swollen conformation triggered by
the increase of temperature was quantified from the difference
between the measured thickness at T4288 K and the initial
value measured at 288 K:
dswðTÞdswð288 KÞ
dswð288 KÞ100 ¼Ddsw
dswð288 KÞ½%(8)
where d
sw
(288 K) is the swollen brush thickness measured
at 288 K and d
sw
(T) the PNIPAM thickness at temperatures
T4288 K. The transition temperature T
tr
was taken as the
temperatureathalfofthebrushcollapse.
Ellipsometry measurements were carried out using a Multi-
scope Null-Ellipsometer from Optrel GbR (Germany). The
instrument is equipped with a red laser (l= 632.8 nm) and a
PCSA (polarizer–compensator–sample-analyzer) setup. For the
measurements in dry condition, a home-built humidity cell was
used, whose inside is flushed by nitrogen stream, reaching a
value of (1.9 0.1)% relative humidity. The angle of incidence
was set to 701and the sample was left drying for 20 minutes
before measurements. For the measurements in liquid, the
sample was soaked in a stainless steel cell filled with the urea
solution. The instrument was equipped with light guides to
drive the incident beam directly at the substrate/water interface
and avoid the reflection at the liquid/air interface. Prior to the
measurements, the sample was swollen in urea solution at
288 K for at least 2 h. A thermal cycle was applied by heating the
liquid by means of a copper plate underneath the sample
holder. The temperature of the liquid environment was measured
continuously with a precision of DT=0.01 K by means of a
temperature sensor immersed in the sample cell.
2.4 Simulation details
A PNIPAM molecule with 24 monomers was studied by atomistic
molecular dynamics (MD) simulations with the GROMACS
4.6.2 software package.
58–60
The force field parameters for
PNIPAM presented in ref. 35 have been used to guarantee the
occurrence of the coil-to-globule transition at temperatures
below 328 K
34,35
while generalized AMBER force fields were
used for urea.
61,62
Typical swollen and collapsed PNIPAM
configurations have been obtained by using the original Meta-
dynamics approach
63–65
as implemented within the PLUMED
package.
66
We extracted the most swollen (radius of gyration
R
g
= 1.4 nm) and the collapsed state of PNIPAM (radius of
gyration R
g
= 0.8 nm) as found during the Metadynamics
simulation and inserted them into cubic simulation boxes to
study the influence of pure water, low and high aqueous urea
PCCP Paper
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
This journal is ©the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 | 5327
concentrations, respectively. The position of the backbone C
a
carbon atoms have been kept fixed by using position restraints.
We randomly inserted the corresponding number of urea and
TIP3P water molecules
67
to model the required urea concentra-
tions. In addition, we also simulated a PNIPAM molecule in
pure water as a reference system. Electrostatic interactions for
all systems have been calculated by the Particle Mesh Ewald
method
68
and all bonds have been constrained by the LINCS
algorithm.
69
First, we performed an energy minimization
followed by a 2 ns warm-up run in a NPT ensemble at 288 K
and 328 K with the Berendsen barostat and thermostat,
70
followed by NPT simulations of 20 ns for the production run
with the same parameter sets. The minimum and maximum
values for the length of the cubic simulation box are given by
hli
min
= 4.7 nm and hli
max
= 6.49 nm in the NPT simulation
depending on the system and the temperature. The corres-
ponding effective urea concentrations for the different systems
can be found in Table 1.
Hydrogen bonds were defined by a maximum distance
criterion of 0.35 nm between acceptor and donor pairs with a
maximum angle of 351.
In fact, the study of polymer brushes is a challenging task
for atomistic simulations. For the minimization of finite-size
effects, a large number of grafted PNIPAM chains have to be
considered which limits the applicability of atomistic approaches.
In contrast, insights into polymer brush behavior can be provided
by computationally efficient coarse-grained (CG) simulations
71
in
which specific chemical details areusuallyneglected.Duetothese
reasons, CG approaches are not suitable for the detailed study of
the PNIPAM–urea interactions such that we decided to simulate
single PNIPAM molecules in an atomistic approach. The usage
of restrained PNIPAM configurations was motivated by recent
publications.
27,40
In fact, the simulation of PNIPAM coil-to-globule
changes is a challenging task which mightresultinaninsufficient
sampling accuracy with regard to the interpretation of conforma-
tional transitions as rare events whose timescale usually exceeds
the simulated time interval. Furthermore, the direct evaluation of
free energy landscapes by free energy methods like metadynamics
or umbrella sampling might also lead to wrong results due to
inappropriate reaction coordinates and the presence of hysteresis
effects.
72
Thus, the usage of restrained PNIPAM conformations
as reference states avoids these drawbacks and provides a
meaningful interpretation of the observed effects.
27,40
2.5 Implications of the Kirkwood–Buff theory and local/bulk
partition coefficients
The Kirkwood–Buff (KB) integral is given by
Gab ¼4pð1
0
r2gmVT
ab ðrÞ1
dr(9)
in the limit of infinite distances with the radial distribution
function g
ab
(r) between molecular species aand b.
16,21,73–78
Since the full integration to infinite distances is not applicable
for radial distribution functions obtained by computer simula-
tions, the introduction of a cut-off radius is necessary.
76,79,80
Furthermore, as the Kirkwood–Buff integrals can be also iden-
tically evaluated in the NpT or NVT ensemble,
74,76
the equation
above can be rewritten as
Gab rc
ðÞ4pðrc
0
r2gNpT
ab ðrÞ1
dr(10)
with the cut-off radius r
c
.
77,79,80
The value of r
c
= 1.8 nm was
used for the data analysis, since the results of the radial
distribution functions showed converged values from this point
onwards. The physical meaning of Kirkwood–Buff integrals can
be interpreted as the excess volume of a co-solute baround the
central particle of a solute a, and they can be also used to study
the local solvent accumulation behavior. In terms of notation,
in the following description the solvent (water) will be denoted
by the subscript a,b= ‘1’, the solute (PNIPAM) by ‘2’ and the
osmolyte (urea) by ‘3’.
The preferential binding coefficient n
23
between PNIPAM
and urea can be obtained from the difference of the KB
integrals describing the distribution of water and urea around
a PNIPAM chain, according to
n
23
=r
3
(G
23
G
21
) (11)
where r
3
denotes the urea bulk number density. Note that the
argument r
c
for the KB integrals has been omitted. The pre-
ferential binding coefficient is connected with the transfer
free energy
DF
23
=RTn
23
(12)
with Rthe molar gas constant and Tthe temperature. The value
for the transfer free energy estimates the amount of free energy
which is needed to transfer a co-solute from the bulk to the
surface of the solute.
The effects of osmolytes on macromolecular conformations
can be evaluated in terms of the chemical equilibrium constant
K=p
s
/p
c
where p
s
and p
c
denote the fraction of PNIPAM
molecules in the swollen (s) and the collapsed state (c). From
the relation
@ln K
@ln a3
¼Dn23 ¼ns
23 nc
23 (13)
the chemical activity of urea a
3
and the chemical equilibrium
constant can be associated with the preferential binding
Table 1 Average effective urea concentrations c
u
for different restrained
PNIPAM configurations defined by specific radii of gyration R
g
and tem-
peratures Tin the NPT simulations. The following abbreviations are used
to define the different combinations of urea concentration, polymer
conformation and temperature: (l)ow or (h)igh urea concentration, (s)wollen
or (c)ollapsed conformation, (288) and (328) for the simulation temperature
T[K] R
g
[nm] c
u
[mol L
1
] Conformation Label
288 0.8 0.5 Collapsed lc288
328 0.8 0.5 Collapsed lc328
288 1.4 0.5 Swollen ls288
328 1.4 0.5 Swollen ls328
288 0.8 6.1 Collapsed hc288
328 0.8 4.8 Collapsed hc328
288 1.4 6.1 Swollen hs288
328 1.4 4.8 Swollen hs328
Paper PCCP
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
5328 |Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 This journal is ©the Owner Societies 2016
coefficients of urea to the swollen and the collapsed PNIPAM
conformation (n
s
23
for the swollen and n
c
23
for the collapsed
conformation).
76
The combination of eqn (13) with eqn (12)
allows us to connect the chemical equilibrium in presence of
osmolytes with the difference in the transfer free energy DDF
23
,
DDF
23
=DF
s
23
DF
c
23
(14)
to yield the final relation
DDF23 ¼RT @ln K
@ln a3
(15)
In accordance with the definition of the reaction constant K,it
can be concluded that a change of the free energy can be
associated with a change of the temperature if it is assumed
that the free energy is mostly dominated by enthalpic contri-
butions. Moreover, with regard to eqn (13), it can be also
concluded, that the chemical equilibrium is shifted to the
conformation with a stronger osmolyte accumulation.
Another robust parameter to study the osmolyte accumula-
tion behavior is given by the local/bulk partition coefficient
40,81
according to
KpðrÞ¼ nuðrÞ
hi=nwðrÞ
hi
ðÞ
ntot
untot
w
(16)
where hn
X
(r)idenotes the average number of water (X = w) or urea
molecules (X = u) within a distance rand n
tot
X
the total number of
urea or water molecules in the simulation box. Thus, a prefer-
ential exclusion behavior leads to K
p
(r)o1 and a preferential
binding of the osmolyte to K
p
(r)41atshortdistancesr.
Considering the local/bulk partition coefficient around the
swollen PNIPAM configuration K
s
p
(r) = exp(DF
s
p
(r)/RT) and
around the collapsed configuration K
c
p
(r) = exp(DF
c
p
(r)/RT),
respectively, the local/bulk partition coefficient free energy
difference can be expressed by
DDFKpðrÞ¼RT log Ks
pðrÞ
Kc
pðrÞ
! (17)
Eqn (17) implies that a negative value of DDF
K
p
(r) indicates a
stronger accumulation of urea around the swollen state, and
therefore the shift of the chemical equilibrium towards chain
swelling, whereas a positive value reveals the stabilization of the
collapsed conformation due to a stronger urea accumulation
around the collapsed chain. Herewith, we exactly follow the
implications given by eqn (13). Noteworthy, it can be also seen
that even for negative preferential binding coefficients (eqn (11))
or local/bulk partition coefficients K
p
(r)41, a shift of the
chemical equilibrium for DDF
K
p
(r)a0 at short distances rcan
be observed, which is purely induced by the accumulation
behavior of urea.
3 Results
3.1 Experimental results
The relative change of ellipsometric thickness Dd
sw
/d
sw
(288 K)
calculated according to eqn (8) is reported in Fig. 1 as a
function of temperature for different urea concentrations (c
u
).
Two important features in the obtained trends can be noticed:
a shift of T
tr
to lower values for increasing urea concentration
c
u
as denoted by the left arrow, and a weaker brush collapse in
urea solutions compared to pure water as denoted by the right
arrow. The weaker brush collapse above the transition tem-
perature at high urea concentrations can be discussed with the
arguments given by Cremer and co-workers in ref. 38, where
increasing PNIPAM radii observed at increasing urea concen-
tration were explained by bridging effects of the osmolyte in
order to cross-link PNIPAM chains. An analogous effect in this
case might be responsible for the reduced brush collapse at
high c
u
. In order to analyze the effects of urea on the thermal
behavior of PNIPAM in more detail, T
tr
and f
sw
were extracted
from the ellipsometry curves. The values of T
tr
for PNIPAM
brushes in presence of different urea concentrations are pre-
sented in Fig. 2. A systematic decrease of T
tr
for increasing urea
concentrations was found, with the evidence of two distinct
regimes: a low concentration regime between 0 and 0.5 mol L
1
,
with a linear decrease of T
tr
as a function of c
u
(inset in Fig. 2),
and a high urea concentration regime between 2 and 7 mol L
1
with a weaker, but constant decrease of T
tr
. Two distinct regimes,
although not so noticeable, were found also for single PNIPAM
chains.
38
Thus, it can be concluded that this effect is not
exclusively characteristic for PNIPAM brushes and it demonstrates
that molecular PNIPAM–urea interactions dominate the chain
behavior, independent of the system geometry. This allows us to
apply the knowledge of single chain–urea interactions to more
complex and crowded molecular systems, like polymer brushes,
which might be of paramount importance for the application of
grafted biomolecular (e.g. proteins) systems.
71
Remarkably, the influence of urea on the transition tem-
perature of PNIPAM may have different origins:
36
on the one
hand, the presence of urea changes the properties of the
aqueous medium;
29
on the other hand, the osmolyte could
Fig. 1 Relative thickness change, Dd
sw
/d
sw
(288 K), as a function of
temperature measured in urea solution at different molar concentration.
The lines are the fit of a sigmoid on the experimental data and are traced as
a guide for the eye. Arrows indicate the effect of added urea.
PCCP Paper
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
This journal is ©the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 | 5329
directly interact with the polymer, as recently demonstrated for
high urea concentrations.
38,82
While at high c
u
the direct urea binding to the polymer is
very likely, it is important to verify which mechanism domi-
nates the change of T
tr
for a PNIPAM brush in presence of a low
molar urea concentration. As a first hypothesis, it is assumed
that urea does not directly interact with PNIPAM, i.e. it is
preferentially excluded from a PNIPAM surface. Under this
condition, the shift of T
tr
might be reasoned by a change of
the PNIPAM hydration properties.
From the decrease of the chemical potential of bulk water
upon addition of urea, and from the equilibrium condition at
T
tr
with identical chemical potentials for H
2
O (bulk) and H
2
O
(brush) phase, the following relation between the temperature
shift DT
tr
and the dehydration enthalpy DH
dehy
can be derived:
DTtr ¼
RT
0
tr
2
DHdehy
wu(18)
where T
0
tr
is the transition temperature in pure water
(c
u
= 0 mol L
1
), DH
dehy
corresponds to the dehydration
enthalpy per mole of water and w
u
denotes the urea–water mole
fraction. The full derivation of this relation is given in the ESI.†
It has to be noted that a similar expression was also derived in
ref. 22.
It is worth to mention that this approach represents a
simplified picture of the system, where the transition enthalpy
of the macromolecule from swollen to collapsed state is omitted.
The validity of eqn (18) to describe the experimental data of Fig. 2
at low c
u
would confirm the dilution effect of bulk water by urea,
i.e. indirect effects. Previous studies on the coil-to-globule transi-
tion of PNIPAM reported calorimetric enthalpy values DH
cal
between 5.5 and 7 kJ mol
1
for a single (NIPAM) repeating unit.
83,84
This can be related to the release of one bound water molecule
from each monomer unit, in agreement with previous experi-
mental results.
83,85
Other literature works report the release of a
much higher number of water molecules per NIPAM unit in bulk
or in gels.
86,87
Nevertheless, in our evaluation only the water
molecules directly bound to the monomer unit via H-bonding
are considered.
Assuming that the same process occurs for the dehydration
of PNIPAM brushes in presence of low molar urea concentra-
tions, where direct urea–PNIPAM interactions are negligible, it
follows that DH
cal
BDH
dehy
and therefore it is possible to
calculate the theoretical trend of DT
tr
as a function of c
u
for the
reported dehydration enthalpies of DH
cal
(1) = 5.5 kJ mol
1
and
DH
cal
(2) = 7 kJ mol
1
. The corresponding functions are shown
as solid lines in the inset of Fig. 2. The agreement between
experimental data and theoretical trends corroborates the
assumption that the dehydration mechanism of PNIPAM in
low concentrated urea solutions is comparable to the dehydration
in pure water in accordance to ref. 83 and 84. Thus, the shift of the
transition temperature can be explained by the dilution of water
by urea as induced by the mole fraction w
u
.
A different mechanism is likely to occur in the high concen-
tration regime, where urea is known to have a significant
influence on the stability of proteins and a direct osmolyte–
polymer binding has been reported in previous studies.
38,82
Indeed, the profile of T
tr
shown in Fig. 2 is very similar to
the results presented by Cremer and coworkers
38
obtained by
Fourier transformed infrared (FTIR) spectroscopy measurements.
The reported FTIR data showed a strong amide band for
PNIPAM in 6 M urea solution, with a major contribution from
CQO(PNIPAM)–H
2
O hydrogen bonds and a second minor one
from CQO(PNIPAM)–NH(urea) bonds. An analogous effect is
likely to dominate the collapse of PNIPAM brushes at c
u
Z2M,
as indicated by our results, where the direct binding of urea
with (NIPAM) monomers might introduce the aggregation of
adjacent chains, such that urea acts as a cross-linking agent.
38
Evidence of direct polymer–osmolyte binding was also found by
Wang et al.
82
for poly(N,N-dimethylacrylamide) (PDMA) and
poly(N,N-diethylacrylamide) (PDEA) brushes by atomic force
microscopy-based single molecule force spectroscopy (SMFS).
The enhanced stiffness of the polymer chains in presence of
2 M and 8 M urea solution was explained by the formation
of hydrogen bonds between the polymer side groups and urea
molecules. Cross-linking of urea via hydrogen bonding was also
reported by Lu et al.
88
to explain urea-induced aggregation of
PNIPAM chains observed by static and dynamic light scattering.
According to Cremer and co-workers,
32,38
a schematic repre-
sentation of the bridging mechanism between PNIPAM chains
in presence of urea is shown in Fig. 3. This aspect will be
discussed in more detail in the next section.
To study the effect of urea on the conformation of grafted
chains, the swelling degree f
sw
of PNIPAM brushes below
(288 K) and above (328 K) the phase transition temperature
was calculated according to eqn (7) for the different urea
concentrations. As it is shown in Fig. 4, a stronger swelling
percentage compared to pure water (c
u
= 0 mol L
1
) is achieved
Fig. 2 Transition temperature T
tr
of PNIPAM brushes as a function of urea
concentration. The samples were subjected to a heating cycle from 288 K
to 328 K. The values T
tr
can be related to the temperature where half of the
brush is collapsed. The inset in the plot reports the values of T
tr
between 0
and 0.5 mol L
1
, while the straight lines describe the calculated trends
according to eqn (18) for the calorimetric enthalpy DH
cal
(1) = 5.5 kJ mol
1
and DH
cal
(2) = 7 kJ mol
1
reported for the NIPAM dehydration in water.
Paper PCCP
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
5330 |Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 This journal is ©the Owner Societies 2016
for low urea concentrations both at 288 K and 328 K. In
contrast, in the high concentration regime (c
u
Z2 mol L
1
)
f
sw
is lower (288 K) or similar (328 K) to the swelling degree in
pure water. Analogous to the previous assumption, we rule out
a strong accumulation of urea inside the brush. Therefore
indirect effects might be responsible for the observed enhance-
ment of brush swelling at c
u
= 0.5 mol L
1
. More specifically,
steric effects on the chain conformation due to the presence
of urea in the swelling medium might lead to higher chain
stretching. This aspect is analyzed and clarified by the numerical
studies presented in the next section. It is worth to mention that
this effects might not be visible at concentrations below 0.5 M
due to the very low amount of the osmolyte in the swelling
medium. In the case of high c
u
, there are two important
implications: at low temperature, increasing urea concentrations
decrease the brush swelling, likely due to a decrease of water
bonding (lower hydration) to the polymer chain; at high tem-
perature, the presence of urea molecules around the macro-
molecule might be responsible for the stabilization of the
collapsed state. It has to be noticed that the considered
temperatures of 288 K and 328 K are significantly below and
above the transition temperature, such that temperature depen-
dent dehydration effects, which could in principle influence
T
tr
, can be omitted.
An analogous representation of the structural transition of
PNIPAM brushes across T
tr
is given by the percentage of the
total brush collapse (i.e. reached at 328 K), which is reported in
the ESI.†The obtained trend as a function of c
u
demonstrates a
reduced brush collapse for any urea concentration compared to
pure water. While in the high concentration regime this can
be reasonably explained by the reduced water content in the
brush, which would confirm the direct polymer–urea inter-
action reported by other authors,
38
in the low concentration
regime it is likely the result of a more subtle interplay between
hydration properties and bulk effects of urea.
In summary, the experimental results indicate concentration-
dependent properties of urea on transition temperatures and
resulting conformations of PNIPAM brushes above and below
T
tr
. The understanding of the molecular origins of the observed
effects on phase transition and swelling behavior motivated the
numerical studies presented in the next subsection. Herewith, we
mostly focus on the interactions between PNIPAM, water and
urea in the low/high concentration regimes and below/above the
phase transition temperature.
3.2 Numerical results
The interpretation of the experimental results presented in the
previous section suffer of some speculative hypotheses to deduce
a suitable molecular mechanism to explain the observed effects.
This is an intrinsic limitation of most experimental methods,
but as we demonstrated in a previous publication,
89
it can be
overcome when experiments and simulations are interpreted in a
synergistic way. Following this approach, simulation results are
presented to complement the experimental findings and to
validate their interpretation.
Typical snapshots of a collapsed and a swollen PNIPAM
configuration in a low molar urea solution are presented in
Fig. 5. By introducing specific position restraints to fix the
PNIPAM configurations, it was possible to avoid transitions
into metastable states and to obtain reliable and well-sampled
statistically averaged values. A detailed discussion of this
approach is given in the section Simulation details.
In order to study the hydration behavior, the local Kirkwood–
Buff integrals G
21
(r) for PNIPAM and water molecules at 288 K for
c
u
=0.5molL
1
were calculated. The values for G
21
(r)inpurewater
were also estimated for comparison. The results are presented at
the top of Fig. 6. Only small differences between the G
21
(r)inurea
solution and the value in pure water can be observed. Hence, it
becomes clear that low molar urea concentrations lead to a weak
change in the PNIPAM hydration behavior at low temperatures,
as it was also concluded from the experimental results.
Fig. 3 Schematic representation of the urea bridging effect between
adjacent PNIPAM chains occurring in the high concentration regime
(c
u
Z2 M). Urea as a cross-linking agent significantly enhances the stability
of the collapsed state. The image was inspired by ref. 32 and 38.
Fig. 4 Swelling degree f
sw
of PNIPAM brushes at 288 K and 328 K for
different urea concentrations. The parameter f
sw
was calculated accord-
ing to eqn (7).
PCCP Paper
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
This journal is ©the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 | 5331
Changes in the PNIPAM–water interactions in presence of
urea can be easily evaluated considering the differences of the
Kirkwood–Buff integrals according to
DG
21
=G
u
21
G
w
21
(19)
with G
w
21
for pure water and G
u
21
in presence of urea. The corres-
ponding results at r
c
= 1.8 nm for all investigated combinations
of chain conformation, urea concentration and temperature are
shown at the bottom of Fig. 6. Larger values of DG
21
were observed
for all systems at 328 K and in particular for the swollen
conformations (ls328 and hs328). In contrast, a slightly modi-
fied water accumulation behavior is evident for all systems at
288 K. It can be therefore concluded that at low temperature the
presence of urea only weakly perturbs the water structure
around PNIPAM, in good agreement with previous assumptions
in the literature
2,20
where it was discussed that urea can be well
integrated into the water structure network.
To study the direct and local interactions between PNIPAM
and urea or water molecules, the number of hydrogen bonds
was calculated. The corresponding results and further findings
are shown in Fig. 7 and in Table S3 of the ESI.†At low molar
urea concentrations, the number of water–PNIPAM hydrogen
bonds is comparable to pure water (DH-bonds (water)) for both
temperatures (ls288, lc288, ls328 and lc328). This means that
the first solvent shell around PNIPAM at low c
u
has a similar
composition as in pure water, and only a small number of urea–
PNIPAM hydrogen bonds (H-bonds (urea)) are formed. These
results validate the previous assumptions of a minor PNIPAM–
urea interaction in presence of low c
u
, and support the hypothesis
of indirect effects from the water–urea network on the chain
conformation at low c
u
.
In contrast, a significant decrease of water hydrogen bonds
compared to pure water (DH-bonds (water)) is found at high c
u
(hs288, hc288, hs328 and hc328), and also the total number of
hydrogen bonds is smaller compared with pure water (i.e. large
negative values of DH-bonds (total)). Interestingly, at high
temperature (hs328 and hc328) the number of urea hydrogen
bonds even exceeds the number of water hydrogen bonds
(H-bonds (urea) 4H-bonds (water)) meaning that in this condi-
tion urea replaces water at molecular interfaces.
88
Moreover, from
the data reported in Table S3 of the ESI†itcanbeseenthaturea
hydrogen bonds are energetically stronger than water hydrogen
bonds. From these results it can be concluded that at high c
u
urea
binding dominates the behavior of PNIPAM chains across the
phase transition, while the effects arising from the change of
PNIPAM–water interactions are negligible.
In order to classify the hydrogen bonds with respect to their
related energetic contributions according to the transition state
theory with the Luzar–Chandler approach,
90,91
it is possible to
calculate the forward rate constants kof hydrogen bonds via
kBexp(DF*/k
B
T), where DF* denotes the activation free
binding energy. The corresponding values for water–PNIPAM
and urea–PNIPAM hydrogen bonds are shown in Table S3 of the
ESI.†The most relevant result is the decrease of the activation
free energies of water for increasing urea concentrations.
The replacement of water–PNIPAM hydrogen bonds with
urea–PNIPAM hydrogen bonds is therefore energetically favorable.
Furthermore, for increasing urea concentration, the strength of
Fig. 5 Typical simulation snapshots of PNIPAM in presence of a low molar
urea solution at 288 K. The snapshot on the left side represents a collapsed
PNIPAM configuration with a radius of gyration of 0.8 nm whereas the right
side shows a swollen configuration with a radius of gyration of 1.4 nm.
Both conformations have been used for the study of the hydration and the
urea binding properties.
Fig. 6 Top: Kirkwood–Buff integrals G
21
(r) for water molecules around
the swollen (R
g
= 1.4 nm: red and upper black line) and the collapsed
PNIPAM configuration (R
g
= 0.8 nm: blue and lower black line) in a 0.5 M
urea solution and pure water at 288 K. The black lines represent the
Kirkwood–Buff integral values around the swollen and the collapsed
configuration in pure water, while the blue and the red line correspond
to the values for water molecules in a 0.5 M urea solution. Bottom: Values
for the Kirkwood–Buff integrals G
21
at r
c
= 1.8 nm for water molecules
around all considered PNIPAM configurations and system parameters
(288 K and 328 K, (h)igh and (l)ow molar urea concentrations, (s)wollen
and (c)ollapsed PNIPAM configuration). The red bars for G
w
21
denote the
values in pure water, the blue bars for G
u
21
represent the values in presence
of urea, and the green bars for DG
21
correspond to the difference in the KB
integrals according to eqn (19).
Paper PCCP
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
5332 |Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 This journal is ©the Owner Societies 2016
water hydrogen bonds is slightly lowered, which facilitates the
release of water molecules from the first solvent shell, in
agreement with the experimental results (Fig. 2). Moreover, the
increasing amount of urea molecules favors PNIPAM dehydration
also due to the replacement of water by urea in the first solvent
shell. These conclusions are additionally supported by the
stagnating values of f
sw
at high urea concentrations (Fig. 4).
In order to evaluate the influence of urea binding on the
shift of the chemical equilibrium towards the swollen or the
collapsed state, the local/bulk partition coefficients were esti-
mated according to eqn (16). The results reported in Fig. 8
prove that the local/bulk partition coefficients K
p
(r) for low c
u
at
both temperatures are smaller than 1 at all distances. This
confirms that urea is preferentially excluded from the PNIPAM
backbone at low concentrations, in perfect agreement with the
small number of urea–PNIPAM hydrogen bonds reported in
Fig. 7. The highly stretched PNIPAM conformations observed
at low urea concentrations (c
u
= 0.5 mol L
1
) in Fig. 4 can be
therefore attributed to the weaker urea exclusion around the
swollen conformation in accordance to eqn (13). Although urea
is very poorly bound to PNIPAM, the interaction with the water
molecules around the macromolecule leads to volume exclusion
effects, which contribute to enhance the chain stretching. The
swollen conformation is therefore more favored compared to the
collapsed state. A slightly stronger urea accumulation at short
distances (rr1 nm) is found for high c
u
, which is more
pronounced at high temperature, and demonstrates the
preferential binding of urea to PNIPAM chains. However, the
preferential exclusion of urea for low c
u
results in a slight
dehydration, as demonstrated by the water accumulation beha-
vior according to the KB integrals presented in Fig. 6 and the
negative values for DH-bonds (water). This finding is in good
agreement with the general explanation for the occurrence of a
coil to globule transition for PNIPAM
33–35
and the shift to lower
transition temperatures. It can be assumed that urea molecules
are preferentially excluded from PNIPAM at low c
u
where it is
energetically unfavorable to replace water with urea molecules.
The situation changes for high c
u
at high temperature, where
a significant dehydration upon phase transition occurs and the
strong binding of urea molecules is energetically favorable
to compensate the loss of the water molecules. These two
mechanisms are able to explain the two different slopes for
the decrease of the transition temperature observed for different
urea concentrations in Fig. 3.
The shift of the chemical equilibrium towards swollen or
collapsed PNIPAM conformations as shown in Fig. 4 can be
rationalized with regard to the local/bulk partition coefficient
free energy difference (eqn (17)). It becomes obvious that only
the high molar urea concentration at 328 K indicates a positive
value of DDF
K
p
E0.2 kJ mol
1
at a PNIPAM distance of
r= 0.5 nm, which validates the energetic stability of the
collapsed PNIPAM conformation. For all the other temperatures
and concentrations, swollen conformations are preferred. Based
on these findings, it can be concluded that the chemical equili-
brium is slightly shifted towards the more swollen conformations
for low c
u
at both temperatures, in agreement with the experi-
mental results (Fig. 4), whereas only high temperatures and high
c
u
induce a shift of the chemical equilibrium towards the
collapsed state. The strong attraction of urea to PNIPAM in high
concentrated solutions and at high temperatures may rationalize
the previously discussed effects for urea in terms of a cross-
linking agent between adjacent PNIPAM chains
27,32,38,40,88
above
a critical urea concentration. The presence of a direct urea
binding can be also assumed with regard to the stagnating values
for f
sw
at c
u
Z2molL
1
which can be rationalized by a fully
saturated urea shell around PNIPAM.
Fig. 7 Number of PNIPAM hydrogen bonds in urea solutions with urea
(H-bonds (urea)), water (H-bonds (water)), total number of hydrogen
bonds (H-bonds (total)) and the difference of H-bonding for the same
configuration and temperature in pure water (DH-bonds (water) and
DH-bonds (total)).
Fig. 8 Local partition coefficient for swollen (R
g
= 1.4 nm) and collapsed
(R
g
= 0.8 nm) PNIPAM conformations in presence of high and low molar
urea concentrations at 288 K (top) and at 328 K (bottom).
PCCP Paper
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
This journal is ©the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 | 5333
4 Summary and conclusions
In this work, the effect of urea on the conformational behavior
of PNIPAM brushes was studied by combining optical experiments
and atomistic molecular dynamics simulations. The reported
results indicated a concentration-dependent binding behavior
between urea and PNIPAM. In the low concentration regime
(c
u
r0.5 mol L
1
), urea is preferentially excluded from the
PNIPAM backbone. Hence, the linear decrease of T
tr
was
rationalized by indirect effects of urea, which influence the
dehydration process at high temperature by dilution of bulk
water. However, despite the low number of urea molecules
around PNIPAM in the first solvent shell, it was found that a
more stretched conformation is stabilized due to a more
favorable urea accumulation around the swollen conformation
and excluded volume effects, as a consequence of the water–
urea network around the macromolecule.
92
In contrast, in the
high concentration regime a preferential binding of urea to
PNIPAM was found at both temperatures. Due to the small
number of water molecules bound to PNIPAM, it can be assumed
that the shift of the transition temperatures at high c
u
is mostly
dominated by direct urea interactions with PNIPAM. Moreover,
the high positive local/bulk partition coefficient of urea at 328 K
supports the hypothesis of urea acting as a crosslinking agent
between adjacent PNIPAM chains.
32,38,88
Thus, two basic mechanisms for different urea concentrations
are proposed: (1) a preferential exclusion of urea from PNIPAM
surface at low molar concentrations. This was rationalized by the
experimental results, which supported a similar dehydration
mechanism at low c
u
as in pure water, proving the absence of a
direct binding; (2) a preferential binding of urea to PNIPAM
above a critical concentration (c
u
Z2molL
1
). This produces a
further decrease of the transition temperature in agreement to
previous findings,
38,82
and a cross-linking effect due to a favor-
able binding between adjacent chains with a similar mechanism
as proposed earlier.
38,88
Although several differences between
numerical and experimental studies exist, for instance the con-
sideration of a single chain in contrast to polymer brushes,
a reasonable agreement between the results was found. This
qualitative coincidence is of crucial importance, as it demon-
strates that the experimental outcomes are strongly influenced by
single chain properties and not by collective effects of the brush.
Specific effects like cross-linking between the polymer chains
which have been reported for high urea concentrations
38,88
are
not detectable by the simulations. However, indirect hints were
found, like the strong accumulation of urea molecules around
PNIPAM chains by a preferential binding mechanism, providing
a reasonable explanation of the experimental observations. With
regard to the two distinct accumulation regimes of urea, it has
to be mentioned that explicit reasons for a similar behavior
have been also recently discussed,
22
and with regard to charged
co-solutes, the presence of specific binding mechanisms for
chaotropic ions was demonstrated.
93,94
In conclusion, the conformational behavior and the transi-
tion temperatures of PNIPAM brushes are strongly affected
by the presence of urea, either by indirect effects or by direct
polymer–osmolyte interactions. This article demonstrates that the
combined results of experiments and simulations point towards
the most reliable methodology to study molecular mechanisms
and their consequences for macromolecular conformations.
Acknowledgements
The authors greatly acknowledge helpful discussions with Anand
Narayanan Krishnamoorthy, Ewa Anna Oprzeska-Zingrebe, Nico
van der Vegt, Francisco Rodriguez-Ropero, Jan Heyda, Dominik
Horinek and Pavel Jungwirth. The authors J. S., J. M. and C. H.
thank the Deutsche Forschungsgemeinschaft through the cluster
of excellence initiative ‘Simulation Technology’ (EXC 310) and the
SFB 716 for financial support. M. A. S. thanks the excellence
cluster ‘‘The Hamburg Centre for Ultrafast Imaging – Structure,
Dynamics, and Control of Matter at the Atomic Scale’’ of the DFG.
The authors S. M. and R. v. K. acknowledge the International
graduate School IRTG 1524 (DFG) for the financial support.
References
1 P. H. Yancey, J. Exp. Biol., 2005, 208, 2819–2830.
2 D. R. Canchi and A. E. Garca, Annu. Rev. Phys. Chem., 2013,
64, 273–293.
3 D. Harries and J. Ro
¨sgen, Methods Cell Biol., 2008, 84,
679–735.
4 R. Politi and D. Harries, Chem. Commun., 2010, 46,
6449–6451.
5 B. J. Bennion and V. Daggett, Proc. Natl. Acad. Sci. U. S. A.,
2003, 100, 5142–5147.
6 R. Zangi, R. Zhou and B. Berne, J. Am. Chem. Soc., 2009, 131,
1535–1541.
7 D. A. Beck, B. J. Bennion, D. O. Alonso and V. Daggett,
Methods Enzymol., 2007, 428, 373–396.
8 A. M. Bhattacharyya and P. Horowitz, J. Biol. Chem., 2000,
275, 14860–14864.
9 A. K. Bhuyan, Biochemistry, 2002, 41, 13386–13394.
10 M.A.Schroer,M.Paulus,C.Jeworrek,C.Krywka,S.Schmacke,
Y.Zhai,D.C.F.Wieland,C.J.Sahle,M.Chimenti,C.A.Royer,
B. Garcia-Moreno, M. Tolan and R. Winter, Biophys. J., 2010,
99, 3430–3437.
11 M. A. Schroer, Y. Zhai, D. C. F. Wieland, C. J. Sahle, J. Nase,
M. Paulus, M. Tolan and R. Winter, Angew. Chem., Int. Ed.,
2011, 50, 11413–11416.
12 J. Smiatek, R. K. Harishchandra, O. Rubner, H.-J. Galla and
A. Heuer, Biophys. Chem., 2012, 160, 62–68.
13 J. Smiatek, R. K. Harishchandra, H.-J. Galla and A. Heuer,
Biophys. Chem., 2013, 180, 102–109.
14 R. K. Harishchandra, S. Wulff, G. Lentzen, T. Neuhaus and
H.-J. Galla, Biophys. Chem., 2010, 150, 37–46.
15 K. D. Collins, G. W. Neilson and J. E. Enderby, Biophys.
Chem., 2007, 128, 95–104.
16 D. Horinek and R. R. Netz, J. Phys. Chem. A, 2011, 115,
6125–6136.
Paper PCCP
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
5334 |Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 This journal is ©the Owner Societies 2016
17 E. Schneck, D. Horinek and R. R. Netz, J. Phys. Chem. B,
2013, 117, 8310–8321.
18 A. Narayanan Krishnamoorthy, C. Holm and J. Smiatek,
J. Phys. Chem. B, 2014, 118, 11613–11621.
19 K. D. Collins, Biophys. J., 1997, 72, 65–76.
20 K. D. Collins, Methods, 2004, 34, 300–311.
21 J. Smiatek, J. Phys. Chem. B, 2014, 118, 771–782.
22 J. Heyda and J. Dzubiella, J. Phys. Chem. B, 2014, 118,
10979–10988.
23 W. Lim, J. Roesgen and S. Englander, Proc. Natl. Acad. Sci.
U. S. A., 2009, 106, 2595–2600.
24 J. Dunbar, H. P. Yennawar, S. Banerjee, J. Luo and
G. K. Farber, Protein Sci., 1997, 6, 1727–1733.
25 E. P. O’Brien, R. I. Dima, B. Brooks and D. Thirumalai, J. Am.
Chem. Soc., 2007, 129, 7346–7353.
26 T. Street, D. Bolen and G. Rose, Proc. Natl. Acad. Sci. U. S. A.,
2006, 103, 17064–17065.
27 F. Rodriguez-Ropero and N. F. A. van der Vegt, Phys. Chem.
Chem. Phys., 2015, 17, 8491–8498.
28 R. Breslow and T. Guo, Proc. Natl. Acad. Sci. U. S. A., 1990,
87, 167–169.
29 S. N. Timasheff, Biochemistry, 1992, 31, 9857–9864.
30 E. I. Tiktopulo, V. N. Uversky, V. B. Lushchik, S. I. Klenin, V. E.
Bychkova and O. B. Ptitsyn, Macromolecules, 1995, 28, 7519–7524.
31 G. Graziano, Int. J. Biol. Macromol., 2000, 27, 89–97.
32 Y. Zhang and P. S. Cremer, Annu. Rev. Phys. Chem., 2010, 61,
63–83.
33 S. A. Deshmukh, S. K. Sankaranarayanan, K. Suthar and
D. C. Mancini, J. Phys. Chem. B, 2012, 116, 2651–2663.
34 H. Du, S. R. Wickramasinghe and X. Qian, J. Phys. Chem. B,
2013, 117, 5090–5101.
35 H. Du, R. Wickramasinghe and X. Qian, J. Phys. Chem. B,
2010, 114, 16594–16604.
36 C. Hofmann and M. Scho
¨nhoff, Colloid Polym. Sci., 2009,
287, 1369–1376.
37 C. H. Hofmann and M. Scho
¨nhoff, Colloid Polym. Sci., 2012,
290, 689–698.
38 L. B. Sagle, Y. Zhang, V. A. Litosh, X. Chen, Y. Cho and
P. S. Cremer, J. Am. Chem. Soc., 2009, 131, 9304–9310.
39 E. A. Algaer and N. F. van der Vegt, J. Phys. Chem. B, 2011,
115, 13781–13787.
40 F. Rodriguez-Ropero and N. F. van der Vegt, J. Phys. Chem. B,
2014, 118, 7327–7334.
41 Y. Gao, J. Yang, Y. Ding and X. Ye, J. Phys. Chem. B, 2014,
118, 9460–9466.
42 Y. Fang, J.-C. Qiang, D.-D. Hu, M.-Z. Wang and Y.-L. Cui,
Colloid Polym. Sci., 2001, 279, 14–21.
43 L. Liu, Y. Shi, C. Liu, T. Wang, G. Liu and G. Zhang, Soft
Matter, 2014, 10, 2856–2862.
44 K. Fuchise, R. Kakuchi, S.-T. Lin, R. Sakai, S.-I. Sato,
T. Satoh, W.-C. Chen and T. Kakuchi, J. Polym. Sci., Part A:
Polym. Chem., 2009, 47, 6259–6268.
45 M. A. C. Stuart, W. T. S. Huck, J. Genzer, M. Mu
¨ller, C. Ober,
M. Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk,
M. Urban, F. Winnik, S. Zauscher, I. Luzinov and S. Minko,
Nat. Mater., 2010, 9, 101–113.
46 J. Gensel, T. Borke, N. P. Pe
´rez, A. Fery, D. V. Andreeva,
E. Betthausen, A. H. E. Mu
¨ller, H. Mo
¨hwald and E. V. Skorb,
Adv. Mater., 2012, 24, 985–989.
47 P. Akkahat and V. P. Hoven, Colloids Surf., B, 2011, 86,
198–205.
48 K. Matyjaszewski and J. Xia, Chem. Rev., 2001, 101, 2921–2990.
49 P. Laurent, G. Souharce, J. Duchet-Rumeau, D. Portinha and
A. Charlot, Soft Matter, 2012, 8, 715–725.
50 L. C. H. Moh, M. D. Losego and P. V. Braun, Langmuir, 2011,
27, 3698–3702.
51 S. Sanjuan, P. Perrin, N. Pantoustier and Y. Tran, Langmuir,
2007, 23, 5769–5778.
52 S. Christau, T. Mo
¨ller, Z. Yenice, J. Genzer and R. von
Klitzing, Langmuir, 2014, 30, 13033–13041.
53 X. Lu, L. Zhang, L. Meng and Y. Liu, Polym. Bull., 2007, 59,
195–206.
54 J. Xia and K. Matyjaszewski, Macromolecules, 1997, 30,
7697–7700.
55 J. B. Theeten and D. E. Aspnes, Annu. Rev. Mater. Sci., 1981,
11, 97–122.
56 Handbook of Ellipsometry, ed. H. G. Tompkins and
E. A. Irene, William Andrew, Inc., Norwich, 2005, vol. 30,
pp. 1–902.
57 J. R. Warren and J. A. Gordon, J. Biol. Chem., 1970, 245,
4097–4104.
58 D. V. D. Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark
and H. J. C. Berendsen, J. Comput. Chem., 2005, 26,
1701–1718.
59 H. J. C. Berendsen, D. van der Spoel and R. van Drunen,
Comput. Phys. Commun., 1995, 91, 43–56.
60 S. Pronk, S. Pa
´ll, R. Schulz, P. Larsson, P. Bjelkmar,
R. Apostolov, M. R. Shirts, J. C. Smith, P. M. Kasson,
D. van der Spoel, B. Hess and E. Lindahl, Bioinformatics,
2013, 29, 845–854.
61 J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman and
D. A. Case, J. Comput. Chem., 2004, 25, 1157–1174.
62 J. Wang, W. Wang, P. A. Kollman and D. A. Case, J. Mol.
Graphics Modell., 2006, 25, 247–260.
63 A. Laio and M. Parrinello, Proc. Natl. Acad. Sci. U. S. A., 2002,
99, 12562–12566.
64 A. Laio and F. L. Gervasio, Rep. Prog. Phys., 2008, 71, 126601.
65 J. Smiatek and A. Heuer, J. Comput. Chem., 2011, 32,
2084–2096.
66 M.Bonomi,D.Branduardi,G.Bussi,C.Camilloni,D.Provasi,
P.Raiteri,D.Donadio,F.Marinelli,F.PietrucciandR.A.
Broglia, Comput. Phys. Commun., 2009, 180, 1961–1972.
67 W. L. Jorgensen, J. Chandrasekhar, J. D. Madura, R. W.
Impey and M. L. Klein, J. Chem. Phys., 1983, 79, 926–935.
68 T. Darden, D. York and L. Pedersen, J. Chem. Phys., 1993, 98,
10089–10092.
69 B. Hess, H. Bekker, H. J. C. Berendsen and J. G. E. M.
Fraaije, J. Comput. Chem., 1997, 18, 1463–1472.
70 H. J. Berendsen, J. P. M. Postma, W. F. van Gunsteren,
A. DiNola and J. Haak, J. Chem. Phys., 1984, 81, 3684–3690.
71 J. Smiatek, A. Heuer, H. Wagner, A. Studer, C. Hentschel
and L. Chi, J. Chem. Phys., 2013, 138, 044904.
PCCP Paper
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
This journal is ©the Owner Societies 2016 Phys. Chem. Chem. Phys., 2016, 18, 5324--5335 | 5335
72 J. Smiatek, D. Janssen-Mu
¨ller, R. Friedrich and A. Heuer,
Physica A, 2014, 394, 136–144.
73 J. G. Kirkwood and F. P. Buff, J. Chem. Phys., 1951, 19,
774–777.
74 A. Y. Ben-Naim, Statistical thermodynamics for chemists and
biochemists, Springer, 1992.
75 B. M. Baynes and B. L. Trout, J. Phys. Chem. B, 2003, 107,
14058–14067.
76 V. Pierce, M. Kang, M. Aburi, S. Weerasinghe and P. E.
Smith, Cell Biochem. Biophys., 2008, 50, 1–22.
77 J. Smiatek, A. Wohlfarth and C. Holm, New J. Phys., 2014,
16, 25001.
78 P. E. Smith, Biophys. J., 2006, 91, 849–856.
79 P. E. Smith, J. Phys. Chem. B, 1999, 103, 525–534.
80 S. Weerasinghe and P. E. Smith, J. Phys. Chem. B, 2003, 107,
3891–3898.
81 E. Courtenay, M. Capp, C. Anderson and M. Record,
Biochemistry, 2000, 39, 4455–4471.
82 C. Wang, W. Shi, W. Zhang, X. Zhang, Y. Katsumoto and
Y. Ozaki, Nano Lett., 2002, 2, 1169–1172.
83 H. Schild and D. Tirrell, J. Phys. Chem., 1990, 94, 4352–4356.
84 I. Bischofberger, D. C. E. Calzolari, P. De Los Rios,
I. Jelezarov and V. Trappe, Sci. Rep., 2014, 4, 4377.
85 H. G. Schild, M. Muthukumar and D. A. Tirrell, Macro-
molecules, 1991, 24, 948–952.
86 Y. Ono and T. Shikata, J. Am. Chem. Soc., 2006, 128,
10030–10031.
87 M. Shibayama, M. Shibayama, S.-Y. Mizutani, S.-Y. Mizutani,
S. Nomura and S. Nomura, Macromolecules, 1996, 29, 2019–2024.
88 Y. Lu, X. Ye, K. Zhou and W. Shi, J. Phys. Chem. B, 2013, 117,
7481–7488.
89 S. Micciulla, P. A. Sa
´nchez, J. Smiatek, B. Qiao, M. Sega,
A. Laschewsky, C. Holm and R. von Klitzing, Soft Mater.,
2014, 12, S14–S21.
90 A. Luzar and D. Chandler, Nature, 1996, 379, 55–57.
91 D. van der Spoel, P. J. van Maaren, P. Larsson and
N. Timneanu, J. Phys. Chem. B, 2006, 110, 4393–4398.
92 A. Soper, E. Castner and A. Luzar, Biophys. Chem., 2003, 105,
649–666.
93 K. B. Rembert, J. Paterova
´, J. Heyda, C. Hilty, P. Jungwirth
and P. S. Cremer, J. Am. Chem. Soc., 2012, 134, 10039–10046.
94 P. Jungwirth and P. S. Cremer, Nat. Chem., 2014, 6, 261–263.
Paper PCCP
Open Access Article. Published on 21 January 2016. Downloaded on 15/05/2017 15:39:41.
This article is licensed under a
Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
View Article Online
